G protein-coupled receptor 37 (GPR37) and GPR37-like 1 (GPR37L1) are orphan G protein- coupled receptors enriched in the central nervous system. However, their cellular distribution in pituitary tissue remains unclear. Here, we mapped Gpr37l1 expression in the adult rat anterior pituitary and identified the expressing cell type(s). Quantitative real-time PCR demonstrated that Gpr37l1 mRNA is significantly more abundant than Gpr37 in the anterior lobe. In situ hybridization (ISH) revealed sparsely distributed Gpr37l1-positive cells throughout the anterior lobe, and the signals did not colocalize with major hormone-producing cell types. Combined ISH and immunohistochemistry further showed that Gpr37l1 mRNA is localized to S100B protein-immunoreactive cells, commonly referred to as folliculo-stellate (FS) cells, and that only a subset of S100B-positive FS cells expresses Gpr37l1. Single-cell RNA-seq analysis corroborated these findings, as Gpr37l1-expressing cells were distributed within the broader S100b-expressing cluster, without forming a distinct subcluster, while showing no detectable Gpr37l1 expression in endocrine lineages or vascular-associated cell populations. Together, these data provide a cell-type-resolved map of Gpr37l1 expression in the adult rat anterior pituitary and identify Gpr37l1 as a marker of heterogeneity within FS cells, establishing a foundation for future studies on GPR37L1 signaling in pituitary microenvironmental regulation.
Astrocytes are the most common glial cells in the mammalian central nervous system. Their diverse structures and functions, along with their critical role as key regulators of brain activity, have recently gained recognition. However, the lack of specific cell markers makes it difficult to identify particular subpopulations. In this study, we examined the distribution of traditional astrocyte markers-glial fibrillary acidic protein (GFAP), SRY-box9 (SOX9), and S100 protein beta (S100β)-in the adult mouse brain. In the cerebral cortex, striatum, and thalamus, most cells did not express GFAP, with limited overlap between GFAP and either SOX9 or S100β. In contrast, the pons-medulla showed a higher rate of co-expression of GFAP and SOX9 compared to the cerebral cortex. In the hippocampus, GFAP was widely expressed and showed significant overlap with SOX9 and S100β. Co-staining of SOX9 and S100β was frequently observed in the cerebral cortex, striatum, thalamus, and midbrain, and S100β-positive cells were more common than SOX9-positive cells. These results reveal regional differences in astrocyte marker expression, highlight the limitations of relying on a single marker like GFAP, and underscore the importance of using multiple markers.
Progressive Supranuclear Palsy (PSP) is a primary 4-repeat tauopathy characterized by progressive motor and cognitive decline. Like other tauopathies, tau misfolding and aggregation are prominent but yield PSP-specific features such as tufted astrocytes and globose neurofibrillary tangles. Emerging evidence suggests that nuclear speckle disassembly and mislocalization of RNA-binding proteins, including serine/arginine repetitive matrix protein 2 (SRRM2), may contribute to disease progression, though SRRM2's role in PSP remains unclear. To assess its association with tau pathology, we examined SRRM2 distribution in midbrain neurons, as well as in astrocytes from both cortical and midbrain regions, using post-mortem immunohistochemistry, immunofluorescence, and 3D reconstruction. In PSP midbrain, neuronal SRRM2 immunoreactivity was markedly elevated compared to controls and co-localized with pTauS396, with >80% overlap; co-localization strongly correlated with SRRM2 abundance (r = 0.9809, p = 0.0191). 3D analysis revealed heterogeneity across cases (PSP-1 to PSP-4) in aggregate morphology, SRRM2 levels, and tau associations. In tufted astrocytes, pTauS396 signals were detected in PSP-1 cortex and PSP-4 midbrain. SRRM2 was absent or faint, yet 3D imaging revealed near-complete SRRM2-pTauS396 co-localization (99% in PSP-1 cortex, 89% in PSP-4 midbrain), regardless of SRRM2 abundance. These findings highlight SRRM2 association with pTauS396 in tangle of PSP.
Microscopy with ultraviolet surface excitation (MUSE) enables rapid fluorescence imaging of tissue surfaces, but MUSE images differ markedly from conventional hematoxylin and eosin images, making cancer delineation challenging for routine pathological practice. We investigated the feasibility of deep learning-based semantic segmentation, which assigns a class label to each pixel, for pixel-wise breast cancer detection in MUSE images. Fresh breast tissues from 30 mastectomy patients with breast cancer were stained with terbium and Hoechst and imaged by MUSE. A total of 150 cancerous images (five per case) were manually annotated into cancerous and non-cancerous classes, and 300 non-cancerous images (ten per case) were collected. Models were trained and evaluated using five-fold nested cross-validation, comparing a cancer-only (CO) model trained solely on cancerous images with a cancer plus non-cancer (CN) model trained on both cancerous and non-cancerous images. The CO model achieved a higher Dice score than the CN model (CO, 0.7478; CN, 0.7343). Sliding window-based majority voting post-processing reduced scattered false-positive areas and improved Dice scores (CO, 0.7984; CN, 0.7849). These results support the feasibility of deep learning-based semantic segmentation for visualizing breast cancer regions and provide a basis for future quantitative applications using MUSE images.
Periodontitis is a chronic inflammatory disease characterized by collagen degradation and alveolar bone loss. Although estrogen contributes to periodontal homeostasis, the role of the membrane-bound G protein-coupled estrogen receptor 30 (GPR30) in periodontitis remains unclear. This study investigated the temporal involvement of GPR30 in ligature-induced periodontitis, focusing on alveolar bone changes, collagen integrity, fibroblast activation, and epigenetic regulation. Twenty male Wistar rats were allocated to control and periodontitis groups and evaluated at days 7, 14, and 21 following placements of a stainless-steel ligature around the maxillary first molars. Alveolar bone loss was assessed radiographically, while inflammatory changes and collagen organization were examined using hematoxylin-eosin and Masson's trichrome staining. Immunohistochemistry was performed to evaluate GPR30, collagen I/III, α-smooth muscle actin (α-SMA), and histone modifications. Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. Alveolar bone loss and collagen degradation were detectable on day 7 and peaked at day 14. GPR30 expression increased during the active inflammatory phase, accompanied by elevated α-SMA and histone H4 lysine 8 crotonylation (H4K8cr), and declined by day 21. Fibroblast transiently adopted a myofibroblast phenotype during inflammation and regressed during early tissue repair. Temporal changes in H4K8cr closely paralleled GPR30 expression. These findings indicate that GPR30 is dynamically associated with inflammatory and remodeling phases of periodontitis and may contribute to epigenetic regulation during periodontal tissue remodeling.
After a crush injury in sciatic nerve fibers, dynamic changes in blood circulation and immune-cell mobilization occur during axonal regeneration. High-resolution visualization under near-physiological conditions is crucial for understanding these mechanisms. Conventional histological techniques introduce perfusion-and dehydration-induced artifacts that obscure circulation. We employed the in vivo cryotechnique (IVCT) to visualize blood flow within sciatic nerve fibers and assess temporal changes during regeneration. In uninjured mice, IVCT preserved native tissue architecture with minimal shrinkage compared to perfusion fixation, with superiority quantitatively shown by fractal analysis. In the crush model, hematoxylineosin, Luxol fast blue, and immunohistochemical staining of IVCT-prepared, freeze-substituted sections revealed axonal degeneration and regrowth. The close association between regenerating fibers and vascular structures, along with erythrocyte distribution, indicates a morphological link between nerves and blood vessels. Electrophysiological assessment using compound muscle action potentials and functional recovery measured by the sciatic functional index demonstrated restored nerve function at 28 days, consistent with histology. These findings suggest that IVCT is a useful method for analyzing peripheral nerve regeneration and vascular dynamics, thereby highlighting its potential as a novel approach in peripheral nerve research.
Lymph nodes (LNs) are critical components of anti-cancer immune responses, orchestrating antigen presentation to T-cells. Aging impairs immune function, but its impact on regional LNs in cancer remains poorly understood. This study investigated age-related morphological and immunological changes in mesenteric LNs and their association with the immune microenvironment in colorectal cancer (CRC). A total of 160 patients who underwent curative resection for CRC were retrospectively analyzed. LN ectasia, defined as sinusoidal dilation accompanied by a reduction in LN sinus macrophages, was evaluated histologically. LN ectasia was significantly more frequent in elderly patients and in right-sided colon cancers. Patients with pronounced LN ectasia showed reduced serum albumin and leukocyte counts. Immunohistochemical analysis revealed that marked LN ectasia was associated with decreased infiltration of CD8+ T cells and Iba1+/CD163+ tumor-associated macrophages in the primary cancer lesion, consistent with a "cold" immune microenvironment. Although LN ectasia was not directly associated with prognosis, it reflected systemic immunosenescence and attenuated local immune activation. These findings suggest that LN ectasia serves as a histological marker of aging-related LN dysfunction, contributing to diminished anti-cancer immunity in CRC.
Cancer cells escape immune surveillance by suppressing immune responses through the binding of Programmed cell Death-Ligand 1 (PD-L1), which is abundantly expressed on the cell surface, to PD-1 on the surface of T cells. The regulation of cell surface PD-L1, one of these immune checkpoint molecules, is extremely important because it is a target for cancer immunotherapy; however, the intracellular trafficking pathway of PD-L1 has not been fully elucidated. Recently, we reported that Rab10, a small GTPase, localizes to a novel tubular endocytic pathway that evades the lysosomal degradation system. In this study, using live cells expressing GFP-PD-L1 and mScarlet-Rab10, we revealed that PD-L1 localizes in Rab10-positive endocytic tubules in some types of cancer cells. Typically, in HeLaM cells, Rab10-positive tubular structures of which membranes have PD-L1 extend from the plasma membrane toward the cell-central region. However, in Rab10-knockout HeLaM cells, no PDL1-localized tubular structures were observed. We also found that PD-L1 dimerized by the PD-L1 inhibitor BMS-202 was removed from the cell surface and Rab10-positive tubular endosomes and transported to the lysosomal degradation system. Taken together, this study provides novel insights that the Rab10-dependent tubular endocytic pathway may play an important role in the intracellular reservoir and recycling of PD-L1 to the surface of cancer cells, possibly regulating the amount of PD-L1 on the cell surface.
Clear cell foci (CCF) are frequently observed in metabolic dysfunction-associated steatohepatitis (MASH) and are considered potential precursor lesions of hepatocyte nuclear factor 1α-inactivated hepatocellular adenoma (H-HCA). To clarify their chronological development, we examined 55 male TSOD mice at 24, 32, 40, and 48 weeks of age using histology and immunohistochemistry for glutamine synthetase (GS), liver fatty acid-binding protein (L-FABP), β-Klotho, and fibroblast growth factor 21 (FGF21). CCF first appeared at 24 weeks and increased markedly with age (from 11% to 81%). All CCF were positive for β-Klotho, and a subset showed FGF21 expression, indicating that CCF represent a hepatocellular state associated with metabolic dysregulation. H-HCA, characterized by GS negativity and reduced L-FABP expression, emerged at 40 weeks and reached an incidence of 29% at 48 weeks. Notably, multiple H-HCA were partially or completely surrounded by β-Klotho-positive CCF, suggesting a morphologic continuum from CCF to H-HCA. Raman spectroscopic analysis demonstrated that CCF exhibit prominent autofluorescence and possess spectral characteristics distinct from both background hepatocytes and tumor tissue, supporting the concept that CCF represent a unique hepatocellular state. These findings indicate that metabolic abnormalities in TSOD mice promote the sequential formation of CCF and H-HCA, establishing this model as a useful platform for studying adenoma development in metabolic liver disease.
Facial nerve palsy profoundly affects facial expression and quality of life, with recovery frequently hindered by aberrant nerve regeneration leading to synkinesis. A comprehensive understanding of three-dimensional (3D) cytoarchitectural organization and adaptive changes within the facial motor nucleus remains constrained by conventional two-dimensional analyses. In this study, retrograde neural tracers (DiI, DiO, Dextran Alexa FluorTM 488/546) were integrated with tissue clearing techniques (CUBIC and LUCID) to reconstruct and analyze the 3D distribution of neurons innervating the orbicularis oris and orbicularis oculi muscles in guinea pigs. Cleared brainstem tissues were imaged using two-photon microscopy, revealing distinct region-specific neuronal clusters within the facial motor nucleus. In a facial nerve injury model, disrupted regional specificity and disorganized neuronal distribution were observed, suggesting misdirected central reinnervation. This novel 3D imaging method enables high-resolution spatial neuronal analysis and reorganization following nerve injury. This approach provides a valuable tool for elucidating facial nerve regeneration and synkinesis, and may facilitate the development of improved therapeutic strategies.
Ampullary carcinoma exhibits marked histological heterogeneity. Although genetic alterations partially account for this diversity, the contribution of epigenetic regulation remains largely unexplored. To elucidate the epigenetic alterations underlying this phenotypic heterogeneity, we investigated methyl-CpG-binding protein 2 (MeCP2) and its downstream target CDX2, and versican (VCAN), a major extracellular matrix proteoglycan implicated in tissue remodeling and tumor-stroma interactions. Seventeen surgically resected cases were analyzed using an integrative approach combining immunohistochemistry, spatial transcriptomics, methylation mapping, electrophoretic mobility shift assays (EMSA), and bioinformatic profiling. In non-neoplastic mucosa, MeCP2 and CDX2 showed reciprocal nuclear expression, a relationship partially preserved in differentiated adenocarcinomas. Spatial transcriptomics identified VCAN as a key MeCP2-associated target gene. Unexpectedly, VCAN, although detectable in MeCP2-negative carcinoma cells, was abundantly expressed in MeCP2-positive cancer-associated fibroblasts (CAFs). Notably, such transcriptional activation by MeCP2, rather than repression, has been reported in neural tissue by previous studies, indicating a conserved mechanism of context-dependent gene regulation. EMSA further demonstrated that hydroxymethylated CpG sites within the VCAN promoter specifically recruited MeCP2, which interacted with CREB to activate VCAN transcription. These findings reveal a dual role of MeCP2: its loss contributes to epithelial heterogeneity, whereas its retained function in CAFs promotes stromal remodeling through VCAN activation.
DNA methylation is a key epigenetic modification that regulates transcriptional activity and is frequently altered during carcinogenesis. To elucidate how cytosine methylation becomes selectively localized to CpG loci during tumor development, we analyzed sequential renal lesions induced by streptozotocin (STZ) in rats. Using microdissection combined with an agarose-bead-based bisulfite sequencing method, the methylation status of the p16Ink4a promoter region was examined from early Armanni-Ebstein lesions through renal cell carcinomas. In early and advanced tubular lesions, cytosine methylation occurred non-selectively at both CpG and non-CpG (CH) sites, whereas in small renal tumors and carcinomas, methylation was progressively restricted to CpG loci. Immunohistochemistry demonstrated strong nuclear expression of p16Ink4a in early lesions, suggesting that active transcription persisted at least until this early stage before the onset of epigenetic silencing. Concomitant expression of Dnmt3b and Dnmt1 was observed, supporting their cooperative involvement in de novo and maintenance methylation, respectively. Notably, the analyzed region overlapped with a CpG-rich segment within the gene body of the alternatively spliced p14Arf gene, which completely fulfill the structural prerequisites for Dnmt3b-mediated methylation. In conclusion, our findings suggest that Dnmt3b-dependent de novo methylation is preferentially initiated within transcriptionally active, CpG-rich regions and subsequently refined to discrete CpG loci as neoplastic transformation advances. This stepwise process provides a mechanistic basis for the selective accumulation of DNA methylation during tumorigenesis and highlights its potential utility as an early epigenetic biomarker of cancer development.
The Hedgehog (Hh) signaling pathway is essential for organ development and tissue homeostasis; its dysregulation is associated with various congenital anomalies and human diseases. Hedgehog-interacting protein 1 (Hhip1) functions as a membrane-bound negative regulator of Hh signaling by directly binding to Hh ligands and limiting their activity in target tissues. While global knockout models have demonstrated the importance of Hhip1 during embryonic development, perinatal lethality has precluded investigations into its postnatal functions. To overcome this limitation, a novel conditional allele of Hhip1 (hereafter referred to as the Hhip1flox or floxed allele, in which exon 2 is flanked by loxP sites) was generated using CRISPR/Cas9-based gene targeting. Cre-mediated excision of exon 2 produced a conditional null allele (Hhip1ex2Δ ) with a frameshift mutation that annulled the Hedgehog ligand-binding domain. Germline recombination through CMV-Cre recapitulated the known pulmonary defects of global Hhip1 knockout mice, validating the model's functionality. Furthermore, using Prx1-Cre to generate limb-specific Hhip1 conditional knockout (cKO) mice, a previously unrecognized role for Hhip1 in maintaining postnatal growth plate architecture was detected. cKO mice exhibited progressive growth plate expansion and long bone overgrowth, together with sustained upregulation of Gli1 expression. These findings establish the Hhip1flox model as a powerful tool for tissue- and stage-specific functional studies of Hhip1 and provide new insights into the spatiotemporal regulation of Hedgehog signaling in development and disease. Importantly, this model offers broad utility for dissecting Hedgehog signaling mechanisms across diverse biological contexts and disease models.
Eribulin, a microtubule inhibitor, is effective as later-line therapy for metastatic breast cancer (MBC) and has been reported to remodel the tumor microenvironment and inhibit epithelial-mesenchymal transition (EMT). However, the association between pretreatment EMT status and eribulin efficacy remains unclear. We retrospectively analyzed 41 patients with MBC (excluding invasive lobular carcinoma) treated with eribulin between 2013 and 2020. Formalin-fixed, paraffin-embedded biopsy specimens were examined by immunohistochemistry (IHC) using anti-E-cadherin (24E10) and anti-vimentin (V9) antibodies. Complete membranous E-cadherin expression (3+) was defined as normal; reduced expression (2+, 1+, 0) as altered. Negative vimentin was considered normal; positive expression, altered. Co-localization of E-cadherin and vimentin was assessed by multi-immunofluorescent staining. Of the 41 patients, 24 responded to eribulin and 17 did not. Progression-free survival (PFS) and overall survival (OS) were significantly longer in responders than in nonresponders (p < 0.001 and p = 0.0044). Altered E-cadherin and/or vimentin expression was more frequently observed in responders (p = 0.013) and associated with longer progression-free survival (p = 0.048). These results suggest that eribulin efficacy may be predicted by altered E-cadherin and vimentin expression before treatment.
In our recent study, we identified Rab10-positive long tubular endosomes, which originate from macropinocytic cups, as a novel endocytic pathway in RAW264 cells. This pathway is unique because it bypasses the lysosomal degradation route and proceeds toward the Golgi region, distinguishing it from previously known endocytic routes. However, its function remains entirely unknown. Upon exploring the cargo transported by Rab10-positive tubular endosomes, we discovered that PD-L1, a cancer immune checkpoint molecule expressed on cancer cells and macrophage surfaces, was abundantly localized in Rab10-positive tubular structures in RAW264 cells. This suggests that PD-L1 may be a significant cargo for these endosomes in macrophages. These findings offer new insights into the role of Rab10-positive tubular endosomes in the intracellular transport of PD-L1, potentially influencing its expression on the cell surface.
Secondary hyperparathyroidism (SHPT) associated with chronic kidney disease (CKD) is characterized by parathyroid hyperplasia, which progresses from diffuse-type to nodular-type lesions. Nodular hyperplasia in SHPT is often considered to exhibit monoclonal proliferation, suggesting a shift toward neoplastic behavior, but the molecular mechanisms underlying this transformation remain poorly defined. In this study, we analyzed 340 surgically resected parathyroid glands from long-term dialysis patients who met clinical indications for parathyroidectomy. Based on histological architecture, lesions were classified into diffuse, nodular, or diffuse-nodular (mixed) hyperplasia. We conducted immunohistochemical analysis of Ki-67 and p16INK4a (CDKN2A), and further assessed region-specific DNA methylation of the p16INK4a promoter using a bisulfite padlock probe method combined with rolling circle amplification. Nodular-type lesions exhibited significantly higher Ki-67 indices and lower p16INK4a expression compared to diffuse-type lesions. In situ methylation analysis revealed increased methylation of the p16INK4a promoter specifically in nodular regions, suggesting epigenetic silencing. Our findings suggest that p16INK4a silencing through promoter methylation may play a critical role in the clonal expansion and histopathological transformation of parathyroid tissue in SHPT. These results underscore the importance of epigenetic regulation in SHPT progression and suggest that p16INK4a methylation could represent a potential biomarker for nodular transformation. The padlock probe-based detection system enabled high-resolution spatial analysis of methylation patterns and may serve as a valuable tool for dissecting epigenetic events in early phase of cellular alterations.
Prostate cancer is one of the most common malignancies in men and remodeling of extracellular collagen, especially collagen type I immensely contributes to the progress of prostate cancer. Discoidin domain receptor 2 (DDR2) is a receptor of collagen type I and transmits intracellular signaling in not only normal cells but also malignant cells, facilitating tumor progression. However, clinical and biological significance of DDR2 has not been well examined in prostate cancer. We therefore immunolocalized DDR2 and collagen type I in 117 prostate carcinoma tissues and correlated their immunoreactivity with clinicopathological characteristics of prostate cancer. We also conducted in vitro experiments using human prostate cancer cell lines to confirm the findings from immunohistochemical study. DDR2 immunoreactivity was positively associated with an aggressive phenotype of prostate cancer, partially in association with dense collagen I tissues which consisted of thin fibers. In addition, DDR2 immunoreactivity was significantly correlated with adverse clinical outcomes of prostate cancer. In vitro experiments revealed that DDR2 promoted proliferation and migration of PC-3 and DU-145 prostate cancer cell lines. It is therefore speculated that DDR2 promoted prostate cancer progression by interacting with collagen I, serving as a potent prognostic factor in prostate cancer.
Sex-determining region Y (Sry) triggers testis development in mammals, and the presence or absence of testicular secretion determines their sex-specific brain phenotype. Mice with Y chromosome replaced by that of Mus domesticus poschiavinus (Y POS ) frequently display sex reversal due to delayed Sry expression. However, brain sexual dimorphism under conditions of disorders of sex development remains unclear. Here, we report sex differences in the sexually dimorphic nucleus of the preoptic area, delineated by cells positive for calbindin D28k, a male-predominant neuronal marker (CALB-SDN), in Y POS mice. The mice were divided into females and males according to gonadal phenotype. Cells immunoreactive (ir) for calbindin D28k (CALB) were more extensively distributed in male Y POS mice, compared with females. The CALB-ir cell numbers in the CALB-SDN were significantly higher in Y POS males than in Y POS females, which had numbers comparable to wild type females. No left-right differences in CALB-ir cell numbers were observed in the CALB-SDN. Collectively, these results demonstrate that sexual dimorphism of the CALB-ir cell cluster in the CALB-SDN strongly correlates with the gonadal sex phenotype rather than with the chromosomal sex in the Y POS mice, suggesting the effect of testicular secretion on the brain sexual differentiation with aberrant Y-linked gene expression.
This study assessed the effectiveness of a combination of platelet-rich plasma (PRP) and β-tricalcium phosphate/polylactic-co-glycolic acid (β-TCP/PLGA) fibers in the treatment of osteoporotic vertebral defects in rats. Seventy-two female Sprague-Dawley rats subjected to ovariectomy to induce osteoporosis were divided into three groups to receive different treatments for critical bone defects created in the lumbar vertebrae. The PRP group received β-TCP/PLGA fibers infused with PRP, the control group received no material, and the other group received the same fibers infused with phosphate-buffered saline (PBS). Over a period of 12 weeks, bone regeneration, macrophage differentiation, and inflammatory responses were evaluated histologically. Compared to the PBS group, the PRP-treated group demonstrated significantly enhanced early stage bone formation, increased expression of osteogenic markers, and a favorable shift in macrophage activity from the M1 inflammatory phenotype to the M2 healing phenotype. These outcomes suggest that the combination of PRP and β-TCP/PLGA fibers not only effectively promotes bone repair under osteoporotic conditions but also modulates the immune response to facilitate better healing, indicating its potential as a beneficial surgical intervention for osteoporotic vertebral fractures.
The long non-coding RNA PVT1 reportedly forms a circular RNA variant (circPVT1). As circPVT1 is expressed in various cancers and has been implicated in promoting cancer cell proliferation and tumor progression, it is considered a potential biomarker and therapeutic target. We previously confirmed that circPVT1 expression varies according to the Gleason pattern, a morphological indicator of malignancy in prostate cancer. In this study, we assessed the expression of circPVT1 using BaseScopeTM assay with prostate cancer tissues and evaluated the correlation with the Grade Group (based on Gleason pattern), an indicator used to morphologically evaluate the degree of malignancy of prostate cancer. The relationship between circPVT1 expression and tumor proliferation was evaluated using cells in which circPVT1 expression was suppressed using the clustered regularly interspaced short palindromic repeats (CRISPR)/RfxCas13d system. BaseScopeTM assay confirmed that circPVT1 expression was significantly higher in Grade Group 2-5 (intermediate- and high-grade groups) than Grade Group 1 (low-grade group). In vitro experiments using the CRISPR/RfxCas13d system showed that specific suppression of circPVT1 expression resulted in a significant reduction in the number of prostate cancer cells. The results of this study suggest that circPVT1 is involved in tumor growth in prostate cancer and may serve as a therapeutic target for moderately and highly malignant prostate cancers that express circPVT1.
